Hemodynamic activity plays an important role in brain function and disease. The connection between neuronal and vascular activity in the brain is poorly understood. This in part stems from the lack of quantitative methods to localize hemodynamic changes in three-dimensions with sufficient temporal and spatial resolution to monitor the progression of the hemodynamic response to a stimulus. Intrinsic Signal Optical Imaging (ISOI) is a camera-based method for imaging cortical activity. ISOI allows researchers to image the hemodynamic changes that accompany neuronal activity by measuring changes in intensity of light remitted from an illuminated brain. Because it is capable of imaging large cortical regions with high spatial and temporal resolution, ISOI has become an important tool for studying the functional architecture and plasticity of the cortex. However, ISOI has two major limitations that have hampered our ability to decipher the complex relationship between hemodynamics and neuronal activity. First, it is often difficult to determine whether measured signals are due to changes in hemoglobin concentration or alterations in hemoglobin oxygenation state. Second, ISOI only provides two-dimensional images, and thus cannot localize hemodynamic activity in depth. We have recently acquired preliminary results using multi-spectral ISOI that demonstrate tomographic localization of hemodynamic activity in rat cortex during whisker stimulation. This is possible because near- infrared light penetrates much more deeply into the brain than visible light. By using many wavelengths of light spanning both the visible and near-infrared (NIR), and applying tomographic imaging methods, we have succeeded in forming three-dimensional tomographic images of hemodynamic activity in the rat somatosensory cortex. In addition, we have also been able to quantify dynamic changes in hemoglobin concentration and oxygenation state by employing spatial frequency domain imaging (SFDI) techniques in conjunction with multi-spectral image analysis. The purpose of this R21 proposal is to validate and refine this exciting new finding. Over the course of the two year funding period, we will incorporate snapshot hyperspectral imaging into our established optical brain imaging methods - ISOI and SFDI. We will optimize our tomographic image reconstruction methods to work in the rat cortex using dynamic tissue simulating phantoms, and validate our approach using our established protocol for whisker stimulation in the adult rat.